The research proposed here concentrates on the regulation of apical membrane Cl channels in airways. In addition a novel genetic approach to the localization of the defective gene on chromosome 7 will take advantage of the screening methods used in the ion transport studies. Cultured cells will be used throughout this proposal. In Project 1 (Widdicombe), levels of second messengers, protein kinases and their target phosphoproteins in normal and CF cells will be compared. Changes in protein phosphorylation will be compared with changes in Cl secretion. In Project 2 (Wine), regulation of Cl channels will be studied using a variety of techniques including patch-clamping. Emphasis will be placed on determining if the same Cl channel defect found in airways can be demonstrated in other affected epithelia, and on whether more than one channel type is affected. In Project 3 (Verkman), regulation of the Cl channel will be studied following reconstitution into liposomes or planar lipid bilayers. Apical membrane vesicles from airway cultures and other tissues will be used as sources of Cl channels. Attempts will be made to purify the Cl channel, though this is not necessary for successful reconstitution. In reconstitution studies, the Cl channel is effectively separated from other apical membrane proteins, providing a direct means of testing whether it is defective in CF. Clinical research will be performed at a clinical/cell acquisition CORE in stanford and human cell culture CORE at each university. The clinical/cell acquisition CORE (Lewiston) will provide airway tissues for the USCF Culture CORE (Finkbeiner). Attempts will be made to transform the cells and to improve the level of differentiation of the cultures as revealed by electrophysiological studies. Ultimately, the clinical/cell acquisition CORE will apply information obtained in the basic science projects to the treatment of patients with CF.